Cataract is the only known disease of the lens. Cataracts are opacities in the lens of the eye that result in a change in refractive index. They are characterized by decreased levels of some of the proteins of the lens. In addition, non-reversible cataracts are characterized by very low levels of the sulfur-containing tripeptide glutathione, .gamma.-L-glutamyl-L-cysteinylglycine (HOOCCH(NH.sub.2)CH.sub.2 CH.sub.2 C(O)NHCH(CH.sub.2 SH)C(O)NHCH.sub.2 COOH). See, W. B. Rathbun, Chapter 14 in Glutathione: Chemical, Biochemical, and Medical Aspects, Vol. III, Part B of the series Coenzymes and Cofactors; D. Dolphin et al., Eds.; Wiley, New York; (1989) pp. 467-510. Glutathione is widely regarded as a major and essential antioxidant for protection of the lens from exogenous noxious agents such as ultraviolet radiation, free radicals, and various toxic compounds of xenobiotic origin.
Biosynthesis of glutathione (GSH) involves two sequential reactions catalyzed by the enzymes .gamma.-glutamylcysteine synthetase and glutathione synthetase (GSH-synthetase) using the three precursor amino acids L-glutamic acid, L-cysteine, and glycine, as shown below: ##STR3##
All substrate-level reactants occur at near enzyme-saturating concentrations in vivo with one exception. This exception is L-cysteine whose cellular concentration is exceedingly low. Therefore, the first reaction in which L-cysteine is required, i.e., the synthesis of .gamma.-L-glutamyl-L-cysteine, is the rate-limiting step of glutathione biosynthesis. Thus, the availability of intracellular L-cysteine is a critical factor in the overall biosynthesis of GSH.
The concentration of GSH in lenses and other tissues generally decreases with age. This is related, at least in part, to the progressive loss of .gamma.-glutamylcysteine synthetase activity and L-cysteine transport capability. Furthermore, the degree of loss of both the biosynthetic enzymes .gamma.-glutamylcysteine synthetase and glutathione synthetase has been shown to be directly proportional to the degree of opacity in human subcapsular cataract. See, for example, W. B. Rathbun and D. L. Murray, Exp. Eye Res., 53, 205-212 (1991); S. S. Sethna and W. B. Rathbun et al., Curr. Eye Res., 2, 735-742 (1983); and W. B. Rathbun et al., Invest. Ophthal. Vis. Sci., 34, 2049-2054 (1993). The concentration of GSH can also be very low in diabetic lenses.
Thus, current efforts are directed at increasing the levels of GSH in the lenses of the aging and diabetic in an attempt to eliminate a key factor for cataractogenesis, as well as increasing GSH levels in other tissues to inhibit oxidative stress and tissue degradation. This has been done by implementing the following therapeutic strategies: (a) providing L-cysteine, the key precursor amino acid; (b) providing the dipeptide precursor .gamma.-L-glutamyl-L-cysteine, thereby by-passing the first biosynthetic step involved in GSH biosynthesis; or (c) providing GSH itself and bypassing both synthetic steps. See, for example, S. M. Deneke et al., Am. J. Physiol., 257, L 163-L 173 (1989). However, exogenously administered L-cysteine and/or GSH are not effective in raising cellular GSH levels due to their rapid catabolism and/or poor transport into the cells.
The cysteine prodrugs, L-2-oxothiazolidine-4-carboxylic acid, and ribosecysteine, the latter a thiazolidine-4-carboxylic acid derived from the condensation of D-ribose with L-cysteine, are effective cysteine delivery systems that raise cellular glutathione levels. See, J. M. Williamson et al., Proc. Natl. Acad. Sci., USA, 78, 936-939 (1981); J. C. Roberts and H. T. Nagasawa et al., J. Med. Chem., 30, 1891-1896 (1987); and A. M. Holleschau and W. B. Rathbun et al., Lens Res., 3, 107-117 (1986). These thiazolidine-4-carboxylic acids have been claimed to delay cataract formation in rats fed an elevated sugar diet (W. H. Garner et al., Allergan, Inc., Eur. Pat. Appln. No. EP 373,002, Jun. 13, 1990). However, L-2-oxothiazolidine-4-carboxylic acid requires pyroglutamyl hydrolase (5-oxoprolinase) to release cysteine. This enzyme is low in lens (V. N. Reddy, et al., Invest. Opthal., 14, 228-232 (1975)). Furthermore, ribose-cysteine dissociates non-enzymatically in aqueous solutions (J. C. Roberts et al., Med. Chem. Rev., 1, 213-219 (1991)) thereby rendering it difficult to prepare stable aqueous pharmaceutical dosage forms.
The dipeptide prodrug of glutathione, .gamma.-L-glutamyl-L-cysteine monomethyl ester (methyl ester group on the cysteinyl moiety), has been shown to raise cellular GSI-I levels by effectively by-passing the .gamma.-glutamylcysteine synthetase-catalyzed reaction. See, A. Ohtsu et al., Japanese Patent No. 64-26516 (1989); and A. Ohtsu et al., Ophthalmic Res., 23, 51-58 (1991). The GSH prodrug glutathione monoethyl ester (ethyl ester group on the terminal glycine) is readily transported across cell membranes and hydrolyzed by intracellular esterases to release the GSH directly. Furthermore, a series of methyl, ethyl, n-propyl, and isopropyl mono esters of GSH as well as methyl, ethyl, n-propyl, and n-butyl diesters of GSH have also been prepared as lipophilic prodrug forms of GSH. See, M. E. Anderson et al., Anal. Biochem., 183, 16-20 (1989). However, aqueous pharmaceutical formulations of these mono and diesters are unstable and rapidly deteriorate over time. Thus, what is needed are new, stable prodrug forms capable of increasing the cellular concentration of glutathione, which is beneficial, for example, in the treatment and/or prevention of cataracts and oxidative stress and tissue degradation.